Our approach to the complex problem of ribosome self-assembly is two complementary lines of experimentation. First we intend to examine the role that 16S RNA conformational transitions play in the process of 30S ribosome reconstitution. Previous studies from our laboratory have shown that different RNA conformational states can be detected by essentially two independent techniques. The first technique employs a sensitive gel electrophoresis system which physically separates subtle RNA conformors. We have shown that the RNA physically goes through several conformational changes during the self-assembly reaction. In this proposal we would like to determine precisely which ribosomal proteins are responsible for these alterations in RNA structure during assembly. The second technique, employed previously, relies on our observation that different preparations of 16S RNA can specifically bind different individual ribosomal proteins. Using this approach we have shown that the binding of one protein to the RNA in some cases induces a change in the structure of the RNA which allows it to bind specifically a second protein. We would like to examine most or all of the classical assembly map relationships to see which reflect protein dependent, induced conformational changes in the RNA. Our second line of investigation will attempt to develop techniques which will permit us to determine the precise roles various portions of individual proteins play in assembly. We intend to prepare fragments of individual ribosomal proteins by chemical and enzymatic methods. Large fragments will then be surveyed for their ability to participate in 30S self-assembly. If the fragment of a given protein can become a member of the reconstituted complex, we shall then be able to determine whether the missing region of that protein has any detectable effect on the ability of other proteins to become incorporated. Such information, along with a detailed model of protein relationships derived from immunoelectron microscopy and with a knowledge of what portion of the protein is lost by the fragmentation relative to the amino acid sequence, should allow us to tentatively suggest specific orientations of the elongated proteins.